1. Field of the Invention
The present invention relates to a method for examining a gene which is useful for the detection and identification of a causative fungus of disease attributed to infection and to a kit for gene examination.
2. Description of the Related Art
Examples of infection caused by fungi include skin disease and deep-seated mycosis.
Meanwhile, some skin diseases cause itching and smallpox in the skin. The causes of these diseases are impossible to visually identify. In general, these skin diseases may be caused by Trichophyton (athlete's foot) or may be caused by yeast-like fungi such as Candida. These skin diseases are infections caused by fungi. Alternatively, skin disease may be caused not by such fungi but by herpes viruses or may be atopic dermatitis. A drug to be administered totally differs depending on such a different cause of skin disease. Under these circumstances, the development of a method has been demanded by which the cause of skin disease is rapidly diagnosed by determining whether the skin disease is attributed to fungi, bacteria or viruses or attributed to, among the fungi, filamentous fungi or yeast-like fungi.
In general, a specimen collected from an affected area is treated with a potassium hydroxide solution and observed under a microscope. If a hypha can be confirmed in this observation, a fungus is determined as the cause of the disease. In addition, a species of the fungus is usually identified by culture.
On the other hand, a method for identifying an organism species performed by the determination of a nucleic acid sequence is known. Japanese Patent Publication No. H08-024600 discloses a method for detecting a fungus in a specimen, including amplifying ribosomal RNA universally held by pathogenic fungi. Alternatively, Japanese Patent No. 3167154 discloses a method for detecting Candida albicans among pathogenic fungi, including amplifying the mitochondrial DNA of this fungus. Likewise, U.S. Pat. No. 5,426,027 discloses primers that can be used in a method including: isolating Candida albicans in blood; amplifying a nucleic acid thereof; and detecting this fungus.
Alternatively, Japanese Patent Application Laid-Open No. 2004-258024 discloses a method for identifying a dermatophyte, including performing antigen-antibody reaction with the surface of an isolated fungus using magnetic beads.
On the other hand, Yasuzawa et al. have reported that a nucleic acid is directly amplified by PCR without isolating a fungus from a nail, and a fungal species thereof is identified from the length or number of fragments of the amplification product treated with a restriction enzyme (Yasuzawa et al., (2005), Japanese Journal of Medical Mycology, Vol. 46, No. 1 (Suppl.), p. 89).
Furthermore, the conventional method for identifying an organism species by the determination of a nucleic acid sequence generally involves separating a fungus with specific antibody-immobilized particles and then detecting a gene, as described in detail in, for example, Japanese Patent Application Laid-Open No. H11-142409. Alternatively, Japanese Patent Application Laid-Open No. 2004-313181 discloses a method capable of collectively detecting plural candidates of causative microorganism species by one experiment using a DNA chip.
Thus, a causative fungal species of skin disease can be identified by such conventional techniques.
However, in the visual identification using a microscope, fungal cells are generally difficult to find in the microscope field of view. It may also be difficult to determine whether a filamentous object that has been found is a hypha of a filamentous fungus, a pseudohypha of a yeast-like fungus or dust. Even if the fungus of interest is confirmed to be a yeast-like fungus or filamentous fungus, a fungal species thereof is often impossible to visually identify.
Moreover, the fungal species identification by culture, which is currently performed as a standard method, usually requires a period as very long as approximately 4 weeks. It is not easy to reliably culture a sample collected from a patient. Therefore, such unsuccessful culture highly probably derives a wrong result (false negative) indicating the absence of a fungus in the sample.
In the skin, microorganisms called indigenous microorganisms are also usually present which do not always cause disease. Only these indigenous microorganisms may be grown selectively. In such a case, a causative fungus of disease cannot correctly be identified.
Furthermore, the conventional method for identifying an organism species by the determination of a nucleic acid sequence generally involves separating a fungus with specific antibody-immobilized particles and then detecting a gene, as described in detail in, for example, Japanese Patent Application Laid-Open No. H11-142409. Specifically, after the isolation operation of a fungus, a nucleic acid is further extracted, and the extracted nucleic acid is further sequenced. Thus, this method requires very complicated operation.
Furthermore, the conventional method for identifying an organism species by the determination of a nucleic acid sequence requires designing a primer set for each fungal species to be identified and then performing an amplification experiment by PCR for each possible fungal species. Therefore, the experiment must be performed plural times corresponding to the number of the possible fungal species. Alternatively, a method for multiplexing PCR using plural primer sets by one experiment has been devised and practiced generally as multiplex PCR. In this multiplex PCR method, the experiment is inevitably complicated. This method also requires, for example, respectively introducing different labels for primers or conducting thermal analysis after PCR and as a result, can simultaneously detect only the limited number of fungal species. Both of such an experiment performed plural times or multiplexing by one experiment may be performed. However, in either case, amplification using primers designed for each candidate fungal species is performed for all the candidates. Therefore, the problem is that the amount of a sample collected must be increased according to the number of candidate fungal species.
Japanese Patent Application Laid-Open No. 2004-313181 discloses a method for solving such problems associated with the determination of a nucleic acid sequence. The document discloses a method capable of collectively detecting plural candidates of causative microorganism species by one experiment using a DNA chip. The method disclosed in Japanese Patent Application Laid-Open No. 2004-313181 is directed to a 16S rRNA region of the causative bacterium. However, fungi do not contain such 16S rRNA. Therefore, this method cannot be used for fungi.
The detection method using antigen-antibody reaction, as described in Japanese Patent Application Laid-Open No. 2004-258024, requires isolating fungal cells from a fungus-containing analyte for removing inhibitors of antigen-antibody reaction. Moreover, this antigen-antibody reaction utilizes an antigen on the surface of the fungus. Thus, it is not easy to prepare an antibody capable of sufficiently distinguishing among, for example, closely related fungi, which have similar surface structures.
The method for identifying a fungal species by PCR-RFLP disclosed in Yasuzawa et al., (2005), Japanese Journal of Medical Mycology, Vol. 46, No. 1 (Suppl.), p. 89 indicates the restriction enzyme treatment of PCR amplification products, by which a cleavage site differing among fungal species is expected. In this method, a fungal species is identified from characteristics such as the number or length of fragments produced depending on a fungal species after restriction enzyme treatment. Plural fungal species to be detected may be present. In such a case, according to this method, restriction enzymes must be selected such that the number and length of fragments produced by restriction enzyme treatment differ among these fungal species. Therefore, the selection of appropriate restriction enzymes is more complicated with increases in the number of fungal species to be detected. In the skin, microorganisms called indigenous microorganisms are also present, as already known, which are not always pathogens of infection. The direct extraction of a nucleic acid without isolating a fungus, as described in the document, highly probably causes the coexistence of the nucleic acid of a pathogen of infection with the nucleic acids of such indigenous microorganisms. In this case, the nucleic acid of interest may be contaminated with the nucleic acids derived from unexpected indigenous microorganisms. Therefore, characteristics such as the number or length of fragments obtained by restriction enzyme treatment lead to unexpected complicated combination. Thus, a fungal species is difficult to identify.